Monolayer cultures of the Y-79 human retinoblastoma cell line were obtained recently by growing the cells on substrata specifically covered with poly-O-lysine and fibronectin. The cells thus attached were studied by light and electron microscopy, immunofluorescence and immunoperoxidase before and after differentiation with various agents, such as dibutyryl-cyclic AMP (dbc-AMP) and sodium butyrate (Nabut). Serum-supplemented and serum-free media were used. Mere attachment of the cells resulted in minor degrees of morphologic differentiation, which was enhanced after the addition of the agents. Round cells with processes, as well as flat, substrate-adherent cells appeared. Specific neuronal and glial markers were expressed by the cells in various conditions. Moreover, ultrastructural characteristics of neuronal, photoreceptor, glial and pigmented cells were observed. The results support differentiation of primitive retinoblastoma cells, such as those of the Y-79 line, into various cell types of normal human retina, i.e. photoreceptor, neuronal, Muller and pigmented epithelial cells. These findings not only support origin of retinoblastoma from a primitive cell of the optic disc, but also offer a system to study cell interactions and tumor cell responses to various therapeutic agents.